Method for evaluating the sensitizing potential of a test compound

ABSTRACT

The invention relates to a method for evaluating the sensitizing potential of a test compound, and to a kit for implementing said method.

The present invention relates to a method for evaluating the sensitizingpotential of a test compound and a kit for implementing said method.

The perfume, cosmetics and pharmacy industries must remain competitiveand effective and continue to regularly offer new products, with theconstraint of complying with the human and environmental safetystandards attached to their use. Contact allergy is one of the majorrisks associated with the use of such products.

Cutaneous contact allergy (or atopic dermatitis) is a major publichealth problem in humans. It represents a serious and restrictiveenvironmental immunotoxic event, whose effects must be anticipated whenmarketing products that could induce it. Skin sensitization and,consequently, the associated allergic manifestation, is the result firstof an interaction of an allergenic molecule with specialized epidermalcells, antigen-presenting cells (Langerhans cells, dendritic cells) andthen, second, their presentation by these cells to CD4⁺ and CD8⁺effector T cells. These T cells are the basis of the allergic andinflammatory reaction. However, the allergens, especially those that canbe present in a fragrance, are small molecules that cannot be recognizeddirectly. To be recognized, they must be associated beforehand with selfproteins. Thus, it is the newly-formed heterodimeric complex in the skinthat will finally be presented to T cells in the proximal lymph nodes.Accordingly, the ability of a chemical molecule (fragrance compound orcosmetic ingredient) to be associated with a protein of the user of thisproduct is a prerequisite for the induction of the consecutivepathological skin reaction. This pathological skin reaction could beirritation, sensitization, or in the majority of cases, both irritationand sensitization.

Irritation is a reversible inflammatory reaction in living tissues bychemical action at the contact site. This is recognized by edemaconsecutive to the influx of fluid to the tissues, redness, heat and/orpain. In response to a chemical attack, keratinocytes of the epidermisand fibroblasts of the dermis are stimulated and release cytokines IL1,TNF alpha, IL6 and IL8, as well as mediators such as prostaglandins(PGE2) into the skin which will initiate the inflammatory response.

The delayed and immediate hypersensitivities that are the basis ofsensitization involve the concept of “memory” which emphasizes theirirreversible nature, unlike irritation. In this case as well, themechanisms occur in two phases:

-   -   the first, called sensitization phase, during which the        antigen/allergen is presented to the immune system and, in        particular, to the T cells that record the molecular signal and        regulate, via the cytokines produced, the other cell populations        involved (B cells, T CD8, endothelial cells, macrophages, mast        cells keratinocytes, etc.);    -   the second phase, called the effector phase, during which        various skin cell populations will act via the chemical        mediators responsible for pathologic disorders. In the case of        immediate hypersensitivity, anaphylactic antibodies such as IgE        are generated and bind mast cells and basophils, leading to the        release of histamine, the main vector for allergic        manifestations. In the case of delayed hypersensitivity, it is        cytotoxic T cells (TCD8) that are responsible for skin lesions        by destroying keratinocytes.

Thus, although from a histological point of view, sensitizing andirritant contact dermatitis are very similar, the immunologicalconsequences analyzed at the cellular level are not necessarily similar.Consequently, it is important to have reliable methodologies fordistinguishing them. An original predictive approach is even morenecessary since currently no clear correlation has been demonstratedbetween a given molecular structure and allergenicity in the broadestsense of term.

So far, animals have been used to identify skin sensitizing moleculesand the LLNA (local lymph node assay), based on the inducedproliferation of lymph node lymphocytes after contact with thesensitizer, has been developed. This test was adopted as “Testingguideline 429” by the Organization for Economic Cooperation andDevelopment (OECD) and is still considered as the standard test fordetermination of a sensitizing chemical agent.

The new European restrictions now require the use of methods that do notuse animals and it is therefore vital to develop alternative methods fordetermining if a new composition or new product is likely to represent arisk for humans due to its sensitizing properties.

The Inventors have shown that in vivo recognition of a substance doesnot occur at the draining lymph node, as is generally accepted, butrather at the tissue where the substance comes into contact with thebody, the skin in the present case. This would explain why somesubstances are sensitizing in one tissue and not in another, as is oftenobserved. It therefore appears that it is the reaction in skin tissuethat sends the message to the allergen-presenting cells: the dendriticcells will then transmit it to the T cells in the draining lymph node.

It is generally accepted that skin models are not sufficient to analyzesensitizing responses and it is necessary to have dendritic cells (EP0857971).

The Inventors have now demonstrated that skin constitutes a sufficientmodel to show specific biomarkers for sensitization and/or irritation inhumans and in mice, and that it is not necessary to add other types ofcells if the identified genes are analyzed at a specific time. TheEPISKIN model can thus be the standard tissue for evaluating thesensitizing nature of a test compound.

Moreover, the Inventors have shown that it was not sufficient to show anoverexpression of only one of said biomarkers to conclude thesensitizing potential of a test compound, and that only studying atleast six specific markers for sensitization would allow drawingconclusions about the sensitizing potential of a test compound.

Thus, the present invention relates to a method for evaluating thesensitizing potential of a test compound, comprising the steps of:

a) contacting a test compound with a biological sample;

b) determining the expression level of at least six genes chosen in thegroup consisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S),DAPK2 (death-associated protein kinase 2), FABP4 (fatty acid bindingprotein 4), HSP27 (heat shock 27 kDa protein), IL18 (Interleukin-18),HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1 receptor typeII), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor,alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolate reductase), EHF(ets homologous factor), IVL (involucrin) KRT4 (keratin 4), MELANA(melan-A), NGAL (gelatinase-associated lipocalin), PDZK1IP1 (PDZK1interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2(proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitormember 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1(aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductasefamily 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B,polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin,light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM(glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)Hdehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11),TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family,polypeptide A9, YWHAZ (tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (hemeoxygenase 1), NQ01 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100calcium binding protein A8).

Preferably, said at least six genes are chosen in the group consistingof: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2(death-associated protein kinase 2), FABP4 (fatty acid binding protein4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1(tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1(defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologousfactor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL(gelatinase associated lipocalin), PDZK1IP1 (PDZK1 interacting proteininteraction 1), P13 (peptidase inhibitor 3), PSME2 (proteasome activatorsubunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10(aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductasefamily 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2),CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P(ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1),G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteineligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2),SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxinreductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptideA1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9) andYWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activationprotein, zeta polypeptide),

more preferentially still chosen in the group consisting of: BRAK (CXCchemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associatedprotein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heatshock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein),IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1),CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR(dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin)KRT4 (keratin 4), MELANA (melan-A), NGAL (gelatinase associatedlipocalin), PDZK1IP1 (PDZK1 interacting protein interaction 1), P13(peptidase inhibitor 3 and SERPINB3 (serpin peptidase inhibitor member3),

and even more preferentially still chosen in the group consisting of:BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2(death-associated protein kinase 2), FABP4 (fatty acid binding protein4), HSP27 (heat shock 27 kDa protein) IL18 (IL-18), HSP90 (heat shock 90kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptasealpha/beta 1), or in the group made up of: CXCR1 (interleukin 8receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolatereductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interactingprotein interaction 1), PI3 (peptidase inhibitor 3, PSME2 (proteasomeactivator subunit 2) and SERPINB3 (serpin peptidase inhibitor member 3)

Preferably, the method according to the present invention can alsocomprise another step c) for determining the sensitizing potential of atest compound.

Preferentially, said step c) can consist of a step of selecting saidcompound as presenting a sensitizing potential if the expression levelof at least 6 of said genes is above a threshold value.

Preferably, the method according to the present invention is an in vitromethod. As used here, the term “biological sample” refers to any solidor liquid sample from a subject.

Preferably, said biological sample is a skin sample.

In a particularly preferred manner, the skin sample is a skin samplereconstructed in vitro, such as, for example, the EpiSkin (EPISKIN, LyonFrance), EpiDerm™ (MATEK Corporation, Ashland, Mass.) or SkinEthic™ RHE(SKINETHIC, Nice, France) model. Preferably, said skin samplereconstructed in vitro also comprises a keratin layer.

Even more preferably, the skin sample does not comprise other types ofadditional cells, and more preferentially no additional Langerhanscells.

The test compound can be a compound of various type, structure andorigin, especially a biological compound, chemical compound, synthetic,etc.

The test compound can be any product present in the isolated form ormixed with other products. The test compound can be defined in terms ofstructure and/or composition or can be defined functionally. The testcompound can be, for example, an isolated and structurally definedproduct, an isolated product of undefined structure, a mixture of knownand characterized products or a composition comprising one or moreproducts. One or more compounds can be tested in this way, mixed orseparately.

Such compositions can be, for example, samples of a cosmetic ordermatological product.

Preferably, said test compound is suitable for use on the skin and maybe used in a cosmetic or dermatological composition.

Preferentially, said method allows assessing the sensitizing potentialof a test compound in humans, comprising a step b) of determining theexpression level of at least six genes such as defined in Table 1, andpreferably chosen in the group made up of: CXCR1 (interleukin 8receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolatereductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interactingprotein interaction 1), P13 (peptidase inhibitor 3), PSME2 (proteasomeactivator subunit 2), and SERPINB3 (serpin peptidase inhibitor member3).

“Sensitizing potential” means the risk for the test compound to provokean immunological reaction when contacted with a mammal, preferably ahuman. Thus, sensitizing potential can be considered as the risk ofdeveloping a contact allergy to the test compound.

“Irritant potential” means the risk for the test compound to provoke areversible inflammation of living tissue by chemical action at thecontact site.

Preferably, the method according to the present invention allowsevaluating whether the test compound is likely to induce a contactallergy or atopic dermatitis.

The present invention is particularly suited to identifying a largenumber of compounds. This simple and effective screening can be done ina very brief period of time. In particular, the methods described may bepartially automated, thus allowing effective and simultaneous screeningof diverse and numerous compounds, either in mixed or separate form.

Preferably, in the method according to the present invention, theexpression level of each of said genes is determined by measuring theexpression level of the polypeptides encoded by said gene or a fragmentthereof, or by determining the expression level of the mRNA from saidgene or a fragment thereof.

In one particularly preferred embodiment, the expression level of eachof said at least six genes is determined by analysis of the expressionof mRNA transcripts or mRNA precursors, such as a native RNA, of saidgene. Said analysis can be done by preparing mRNA/cDNA from cells of apatient's biological sample, and hybridizing the mRNA/cDNA with areference polynucleotide. The prepared mRNA/cDNA may be used in analysisby hybridization or amplification that includes, without being limiting,Southern and Northern analysis, PCR (“polymerase chain reaction”), suchas quantitative PCR (Taqman) and the use of probes (“probe arrays”) suchas GeneChip® DNA Matrices® (AFFYMETRIX).

Advantageously, the analysis of the mRNA expression transcript of eachof said at least six genes involves a nucleic acid amplificationprocess, such as, for example, RT-PCR (experimental method described inU.S. Pat. No. 4,683,202), ligase chain reaction (BARANY, Proc. Natl.Acad. Sci. USA, vol. 88, p: 189-193, 1991), self sustained sequencereplication (GUATELLI et al., Proc. Natl. Acad. Sci. USA, vol. 87, p:1874-1878, 1990) and transcriptional amplification system. (KWOH et al.,Proc. Natl. Acad. Sci. USA, vol. 86, p: 1173-1177, 1989), “Q-BetaReplicase” (LIZARDI et al., Biol. Technology, vol. 6, p: 1197, 1988),“rolling circle replication” (U.S. Pat. No. 5,854,033) or any othernucleic acid amplification method, followed by a step of detecting theamplified molecules by techniques well known to the skilled person.These detection modes are particularly useful for detecting nucleic acidmolecules in very small quantities. Thus, according to a preferredembodiment, the method according to the present invention comprises anadditional step of amplifying the mRNA or cDNA of each of said sixgenes, the complementary sequence thereof or a fragment thereof.

Such as used here, amplification primers are defined as being a pair ofnucleic acid molecules that can respectively pair with the 3′ and 5′regions of a gene in a specific manner (positive and negative strands orvice versa) and encompassing a short region of said gene. Generally,amplification primers have a length of 10 to 30 nucleotides and allowamplifying a region of a length comprised between 50 and 200nucleotides. Advantageously the primers used in the present inventionare those listed in Table 1.

In another particularly preferred embodiment, the expression level ofeach of said at least six genes is determined by determining theexpression level of the polypeptide encoded by said gene or a fragmentthereof. Said analysis can be done by using an antibody (for example, aradiolabeled antibody, an antibody labeled with a chromophore, afluorophore or an enzyme) an antibody derivative (for example, anantibody conjugated to a substrate or to a protein, or a ligand of aprotein of a ligand/protein pair (for example biotin-streptavidin)) oran antibody fragment (for example, a single chain antibody, ahypervariable domain of an isolated antibody, etc.) which specificallybinds the polypeptide encoded by said gene.

Said analyses can be done by many techniques known to the skilled personincluding, without being limiting, immunological tests based on the useof enzymatic activity (“enzyme immunoassay” EIA), immunological testsbased on the use of radioactive isotopes (RIA), western blot analysisand ELISA (“enzyme linked immunosorbent assay”) tests.

In the sense of the present invention, “polypeptide” means a sequencecomprising at least two amino acids, and the terms “polypeptide”,“peptide” and “protein” may be used interchangeably.

In the sense of the present invention, “mRNA or cDNA fragment” means asequence of at least 50 nucleic acids, for example at least 100 or 150nucleic acids, preferably at least 200 nucleic acids, for example atleast 250 or 350 nucleic acids, and in a particularly preferred manner,a polypeptide [sic; nucleic acid sequence] of at least 400 nucleicacids.

In the sense of the present invention, “polypeptide fragment” means asequence of at least 50 amino acids, for example at least 100 or 150amino acids, preferably at least 200 amino acids, for example at least250 or 350 amino acids, and in a particularly preferred manner, apolypeptide of at least 400 amino acids.

Preferably, the method according to the present invention also comprisesa step of comparing the expression level of each of said at least sixgenes with a reference value. This reference value can serve as apositive and/or negative control.

A positive control can be conducted, for example, by comparing theexpression level of said at least one gene in the presence of the testcompound with the expression level of said at least one gene in thepresence of a compound known to be sensitizing.

As an example of a compound known to be sensitizing, the following canbe named: 2,4,6-trinitrobenzene sulfonic acid, p-phenylenediamine,dinitrochlorobenzene, benzaldehyde, resorcinol, tetramethylthiuramdisulfide, oxazolone, chloroatranol, diphenylcyclopropenone, potassiumdichromate, cinnamaldehyde, 2-bromo-2-(bromomethyl) glutaronitrile,glyoxal, saccharin, formaldehyde, trimellitic anhydride,methylchloroisothiazolinone, benzyl benzoate, alpha-hexylcinnamaldehyde, eugenol, 2-mercaptobenzothiazole, isoeugenol,diphenylcyclopropenone (DCPP), lauryl gallate (LG),3-3-dimethylaminopropylamine (3-DMAPA), cinnamaldehyde (CA), citral(Cal), 1,4-hydroquinone (HQ), glutaraldehyde (GA),1,2-benzisothiazolin-3-one (Ben), phenylacetaldehyde (PA) and lilial(Li), preferably diphenylcyclopropenone, lauryl gallate,1,4-hydroquinone and glutaraldehyde, and particularly preferably1,4-hydroquinone.

Alternatively, in the present invention, a sensitizing compound can beused as a positive control, such as “fragrance mix”.

A negative control can be conducted in the absence of the test compoundor in the presence of a compound known to be non-sensitizing, such as,for example, olive oil, glycerol, cetyltrimethylammonium bromide (CTAB)and dipropylene glycol.

In the scope of the present invention, we will conclude that a testcompound has a sensitizing potential if an overexpression of said geneis observed with regard to its expression level in the absence of saidtest compound.

“Overexpression” means a significantly higher level of said genecompared to its normal expression level. Preferably, overexpressionmeans an expression level in a biological sample that is greater by atleast 20% than the normal expression level of said gene, preferablygreater by at least 50% than the normal expression level of said gene,and more particularly preferably greater by at least 90% than the normalexpression level of said gene.

“Expression level in the absence of said test compound” or “normallevel” is the expression level of said gene in a control samplepotentially corresponding to the biological sample of a patient who doesnot present sensitization or, preferably, to the mean of the expressionlevel of said gene in different control samples.

Preferably, step b) of said method for evaluating sensitizing potentialcomprises measuring the expression of at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24 and morepreferentially, at least 25 genes chosen in the group made up of thegenes such as defined in Table 1.

Thus, one can conclude that a test compound has a sensitizing potentialif there is an overexpression of at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24 and morepreferentially, at least 25 genes chosen in the group made up of genessuch as defined in Table 1.

In a particularly preferred manner, step b) of said method forevaluating sensitizing potential comprises determining the expression ofthe following group of genes: AKR1B10 (aldo-keto reductase family 1,member B10), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2(aldo-keto reductase family 1, member C2), CYP1B1 (cytochrome P450,family 1, subfamily B, polypeptide 1), FTH1P (ferritin, heavypolypeptide 1), FTL (ferritin, light polypeptide 1), G6PD(glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteine ligasemodifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2), SLC7A11(solute carrier family 7, Member 11), TXNRD1 (thioredoxin reductase 1),UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), UGT1A9(UDP glucuronosyltransferase 1 family, polypeptide A9, YWHAZ (tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, zetapolypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1,subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalyticsubunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)H dehydrogenase,quinone 1) PSME2 (proteasome activator subunit 2), and S100A8 (S100calcium binding protein A8), preferably chosen in the group consistingof: AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1(aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductasefamily 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B,polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin,light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM(glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)Hdehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11),TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family,polypeptide A9, PSME2 (proteasome activator subunit 2), and YWHAZ(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta polypeptide).

In this particularly preferred embodiment, the test compound isdetermined to have sensitizing potential if at least 11 of said genesare overexpressed compared to a reference value.

In a particularly preferred manner, step b) of said method forevaluating sensitizing potential comprises determining the expression ofat least one of the following group of genes: BRAK (CXC chemokine ligand14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2),FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein),IL18 (IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolatereductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interactingprotein interaction 1), PI3 (peptidase inhibitor 3) and SERPINB3 (serpinpeptidase inhibitor member 3),

and even more preferentially, the group of genes BRAK (CXC chemokineligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDaprotein), IL18 (IL-18), IL1R2 (interleukin-1 receptor type II), HSP90(heat shock 90 kDa protein), and TPSAB1 (tryptase alpha/beta 1), or thegroup of genes CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin,beta 1), DHFR (dihydrofolate reductase), EHF (ets homologous factor),IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2),PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3 (peptidaseinhibitor 3) and SERPINB3 (serpin peptidase inhibitor member 3).

In this particularly preferred embodiment, the test compound isdetermined to have sensitizing potential if at least 7 of said genes,preferably at least 8 of said genes are overexpressed compared to areference value.

In one particular embodiment, step b) of said method for evaluating thesensitizing potential of a test compound comprises a step a) ofdetermining the expression level of at least 10, preferably at least 11,12, 13, 14, 15, 16 and still more preferentially of the following groupof genes: AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1(aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductasefamily 1, member C2), CTGF (connective tissue growth factor), CYP1B1(cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P(ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1),G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteineligase modifier subunit), IER3 (immediate early response 3), NQO2(NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7,Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDPglucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDPglucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, zetapolypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1,subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalyticsubunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)H dehydrogenase,quinone 1) and S100A8 (S100 calcium binding protein A8).

and optionally a step β) of measuring the expression level of at least10, preferably 11, 12, 13, 14, 15, 16, 17, 18, 19 and morepreferentially of the following group of genes: BRAK (CXC chemokineligand 14), CTSS (cathepsin S), DAPK2 (death-associated protein kinase2), FABP4 (fatty acid binding protein 4), HSP27 (heat shock 27 kDaprotein), IL18 (IL-18), IL1R2 (interleukin-1 receptor type II), TPSAB1(tryptase alpha/beta 1), HSP90 (heat shock 90 kDa protein), CXCR1(interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR(dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin)KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1interacting protein interaction 1), P13 (peptidase inhibitor 3), PSME2(proteasome activator subunit 2) and SERPINB3 (serpin peptidaseinhibitor member 3), and

a step c) of determining the sensitizing potential of a test compoundwherein the compound is determined to be sensitizing if:

-   -   the expression level of at least 7 genes measured in step α) is        greater than a threshold value; and/or    -   the expression level of at least 7 genes measured in step β) is        greater than a limit value.

Preferably, step b) is done between 2 and 24 hours after step a), stillmore preferably between 4 and 18 hours after step a), preferentiallybetween 5 and 7 hours after step a) and most preferably of all, 6 hoursafter step a).

Another aspect of the invention relates to a method for evaluating thesensitizing power of a test compound, comprising the following steps:

1) obtaining at least one test compound dilution, and

2) determining the sensitizing potential of said test compound at saidat least one dilution by a method such as defined according to any oneof the preceding claims.

“Sensitizing power” means the ability of a given compound to induce asensitization reaction according to the concentration of said compound.Sensitizing power is dependent on the quantity of substance necessary toinduce sensitization. Thus, the lower the sensitizing quantity necessaryto induce a sensitizing response, the stronger the sensitizer is, andvice versa, the higher the sensitizing quantity necessary to induce asensitizing response, the weaker the sensitizer is.

It is thus possible to perform a quantitative analysis of thesensitizing potential of a compound.

Preferably, said test compound is subject to successive dilutions. Thus,steps 1) and 2) will be done for each of the dilutions.

Preferably, said successive dilutions will allow determining the maximumdilution at which said test compound retains sensitizing potential.

Said method for evaluating the sensitizing power of a test compound canalso comprise a step of evaluating the sensitizing power of the testcompound.

Thus, the more a product retains a sensitizing potential aftersuccessive dilutions, the more powerful a sensitizer the test compoundis.

Moreover, the sensitizing power of a test compound will also be afunction of the irritant potential of said test compound. Thus, the factthat a test compound has an irritant potential increases the sensitizingpower of said test compound.

Consequently, in one preferred embodiment, said method also comprises astep of determining the irritant potential of the test compound.

The irritant potential of a test compound can be evaluated by using, forexample, the method described in French patent 1051638.

Thus, one can conclude that a product is extremely sensitizing (ExtremeE), if:

-   -   it has a sensitizing potential at a dilution of 1/1000, and/or    -   it has a sensitizing potential at a dilution of 1/100, but does        not have a sensitizing potential at 1/1000, and has an irritant        potential.

Thus, one can conclude that a product is strongly sensitizing (StrongS), if:

-   -   it has a sensitizing potential at a dilution of 1/100, but does        not have a sensitizing potential at 1/1000, or    -   it has a sensitizing potential at a dilution of 1/10, but does        not have a sensitizing potential at 1/100 and has an irritant        potential.

Thus, one can conclude that a product is moderately sensitizing(Moderate M), if:

-   -   it has a sensitizing potential at a dilution of 1/10, but does        not have a sensitizing potential at 1/100, or    -   it has a sensitizing potential at a dilution of 1/2, but does        not have a sensitizing potential at dilutions below 1/2 and has        an irritant potential.

Thus, one can conclude that a product is weakly sensitizing (weak W) ifit does not have a sensitizing potential at dilutions below 1/2.

According to another aspect, the present invention relates to the use ofat least one, preferably at least 2, at least 3, at least 4, at least 5,at least 6, at least 7 or at least 8 genes chosen in the groupconsisting of: BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2(death-associated protein kinase 2), FABP4 (fatty acid binding protein4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1(tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1(defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologousfactor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL(gelatinase associated lipocalin), PDZK1IP1 (PDZK1 interacting proteininteraction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasome activatorsubunit 2), SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10(aldo-keto reductase family 1, member B10), AKR1C1 (aldo-keto reductasefamily 1, member C1), AKR1C2 (aldo-keto reductase family 1, member C2),CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P(ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1),G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteineligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2),SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxinreductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptideA1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9), andYWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activationprotein, zeta polypeptide) for the in vitro evaluation of thesensitizing potential of a test compound.

The present invention also relates to a kit for the implementation of amethod for in vitro evaluation of the sensitizing potential of a testcompound, comprising means for determining the expression level of atleast six genes selected from the group consisting of BRAK (CXCchemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associatedprotein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heatshock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein),IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1),CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR(dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin)KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2(proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitormember 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1(aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductasefamily 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B,polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin,light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM(glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)Hdehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11),TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family,polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (hemeoxygenase 1), NQ01 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100calcium binding protein A8).

Preferably, said kit will comprise at least six primer pairs eachamplifying at least one gene chosen from the group consisting of: BRAK(CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associatedprotein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heatshock 27 kDa protein), IL18 (IL-18), IL1R2 (interleukin-1 receptor typeII), HSP90 (heat shock 90 kDa protein), TPSAB1 (tryptase alpha/beta 1),CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR(dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin)KRT4 (keratin 4), MELANA (melan-A), NGAL (gelatinase associatedlipocalin), PDZK1IP1 (PDZK1 interacting protein interaction 1), P13(peptidase inhibitor 3), PSME2 (proteasome activator subunit 2),SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-ketoreductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1,member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1(cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P(ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1),G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteineligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2),SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxinreductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptideA1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9),YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activationprotein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochromeP450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteineligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQ01 (NAD(P)Hdehydrogenase, quinone 1) and S100A8 (S100 calcium binding protein A8).

In a particularly preferred manner, said at least one primer pair ischosen in Table 1.

EXAMPLES 1) Demonstration of Biomarkers According to the SkinethicProtocol

Skin pieces of 1.07 cm² were purchased from EP1SKIN. Various substanceswere applied either in the liquid form (30 μL at differentconcentrations) or in the solid form (30 μL PBS or olive oil+30 μg orfewer to assess different powder concentrations) on the skin pieces.After an incubation of 15 min at room temperature, the skin pieces werewashed with PBS (25 mL) and incubated 3 h, 6 h or 18 h at 37° C. in aCO₂ incubator. After incubation, the skin pieces were sampled with apunch and separated from the collagen support. They were then directlyplaced in a “Tri Reagent” solution (Ambion) (1 ml) and immediatelydissociated mechanically.

cDNA Preparation

The tissues were placed in a “Tri Reagent” solution (Ambion) andmechanically crushed. The RNA was prepared according to the protocoldescribed by the supplier with isopropanol precipitation. To prepare thecDNA, the total RNAs were pretreated with DNAse to remove genomic DNAcontaminants. One to 5 μg of total RNA was used for the treatment, withRNAse-free DNAse, RNAsin (1 μL) and random primers (3 μg). Thesuperscript III RT (1.5 mL at 200 U/μL) was then added. The cDNAs werethen tested by RT-PCR.

Quantitative PCR

Real time quantitative PCR was performed using the SYBR Green technologyof Roche LC480 cyclers. The primers were designed to cover theintron-exon junctions to prevent any traces of genomic DNAamplification. Amplification gives amplicons between 100 and 150 bp. Allthe primer pairs were qualified by digestion with restriction enzymesand analyzed by electrophoresis. PCR was performed on 10 μL by using theSybr green 2×PCR mix from Roche in PCR plates from Roche. Target geneexpression was measured after RNA normalization by means of fourhousekeeping genes, and the values are expressed by using the C_(T)method, and expressed in additional expression rates with regard to atheoretical zero (User Bulletin no. 2, Applied Biosystems, December1997).

Results

For this analysis, an application of 15 min followed by washing and apost-incubation of 6 h were done before biopsy and analysis of the genetranscription. The results are shown in Table 1.

TABLE 1expression level of genes specific for sensitivity in the EPISKIN modelExpression Expression level in level in the ab- the pre- sence ofsence of Abbrevi- Gene name Accession sensiting sensiting Sens Antisenseation in French English name number agent agent primer primer BRAK CXC chemokine NM_004887.4 0.65368817 1.8 AAGTACCCGC TTGGTGCTCTGCAGCchimiokine (C-X-C motif) 1 ACTGCGAGGA TTGGGGTGC ligand 14 ligand 14 GAAG(SEQ ID NO: 2) (SEQ ID NO: 1) HSP90 Protéine  Heat shock NM_005348.31.05432172 1.75 TCTGCCTCTGG TTTCCGAAGACGTTC de choc  90 kDa 1 TGATGAGATGCACAAAGGC thermique protein GTT (SEQ ID NO: 82) de 90 kDa (SEQ IDNO: 81) CTSS cathepsins  cathepsin S NM_004079.3 0.51868364 2.29602025TCATACGATC TGGGAACTCTCAGGG S 1 TGGGCATGAA AACTCATCA CCAC (SEQ ID NO: 4)(SEQ ID NO: 3) DAPK2 protéine  death- NM_014326.3 1.01258991 1.45464354TTTCCTTCAGC TGTCACTCTCACAGT kinase associated 5 8 ATCGTGTCCCT TCCTCAGGTassociée protein GT (SEQ ID NO: 6) à la kinase 2 (SEQ ID mort 2 NO: 5)FABP4 Proteine  fatty acid NM_001442.2 0.84442534 2.92074858 ACTGGGCCAGTTTCTGCACATGTAC de  binding 4 1 GAATTTGACG CAGGACACC fixation protein 4, AAGT (SEQ ID NO: 8) des acides adipocyte (SEQ ID gras 4NO: 7) HSP27 Protéine  heat shock NM_001540.3 0.86854312 1.61293690TGCTTCACGC CTGGGATGGTGATCT de choc  27 kDa 7 8 GGAAATACAC CGTTGGACTthermique protein 1 GCT (SEQ ID NO: 10) de 27 kDa (SEQ ID NO: 9) IL18Interleukin Interleukine NM_001562.2 1.19095641 2.30930138 TCATTGACCAAGCCATACCTCTAGG 18 18 8 7 AGGAAATCGG CTGGCTAT (interferon- CCTC(SEQ ID NO: 12) gamma-inducing (SEQ ID factor) NO: 11) IL1R2 Recepteurinterleukin 1 NM_173343.1 1.28062122 1.9 GCCAGCCTTG TTGCGGGTATGAGATtype II de receptor, 8 CAGGAGGACT GAACGGCAG l'interleu- type II CTG(SEQ ID NO: 14) kine 1 (SEQ ID NO: 13) TPSAB1 tryptase tryptaseNM_003294.3 0.04887814 33.9277 ACGGCCCATACT CAGCAGCTGGTCCTG alpha/alpha/beta 1 9 6528 GGATGCACTTCT GTAGTAGA beta 1 (SEQ ID (SEQ ID NO: 16)NO: 15) CXCR1 Recepteur interleukin 8 NM_000634.2 1.44289407 3.49217TATGAATCTGTCC ACCTCATAGCAAACT alpha de receptor, 4 8966 CTGCCCTTCGGACTGGAA l'interleu- alpha (SEQ ID (SEQ ID NO: 18) kin 8 NO: 17) DEFB1defensine, defensin, NM_005218.3 0.66742459 2.43146 TTCCTGAAATCCTAGGCCTGTGAGAAA beta 1 beta 1 7 3704 GGGTGTTGCCT GTTACCACCT (SEQ ID(SEQ ID NO: 20) NO: 19) DHFR dihydro- dihydrofolate NM_000791.31.03301762 2.6 CTCATTTTCTTTC TGCCACCAACTATCC folate reductase 5CAGAAGTCTAG AGACCATGT reductase (SEQ ID (SEQ ID NO: 22) NO: 21) EHFFacteur ets homologous NM_012153.3 0.82749952 2.81578 TTGGCTCTCTCATAGGTATGACACTGTG homologue  factor 7 3657 GTCCTTGGCTT GTAGGTGCT ets(SEQ ID (SEQ ID NO: 24) NO: 23) IVL involucrine involucrin NM_005547.20.94387652 1.85950 TGCCCACAAAGG TCTGGACACTGCGGG 1 5427 GAGAAGTATTGCTGGTTATTT (SEQ ID (SEQ ID NO: 26) NO: 25) KRT4 keratine  keratin 4NM_002272.2 0.92514885 1.35742 TCACATATGTCCC TGCCGGGTGTTGGAG 4 8 071TTCCCAGTCCA AAGTAGTTT (SEQ ID (SEQ ID NO: 28) NO: 27) MELANA melane-Amelan-A NM_005511.1 0.76332759 2.7 TCTTACTGCTCAT TGAAGAGACACTTTG 8CGGCTGTTGGT CTGTCCCGA (SEQ ID (SEQ ID NO: 30) NO: 29) NGAL Gelatinase lipocalin 2 NM_005564.3 1.06935756 1.8 GTGAGCACCAAC AGTTCCGAAGTCAGC des5 TACAACCAGCAT TCCTTGGTT neutro- (SEQ ID (SEQ ID NO: 32) philes NO: 31)PDZK11P1 Protéine PDZK1 NM_005764.3 0.70547940 2.3 CAATCGCCTTTGCACCAGGACTCCATCT d'inter- interacting 4 AGTCAACCACT GCCTTGTTT actionprotein 1 (SEQ ID (SEQ ID NO: 34) PDZK1 NO: 33) PI3 Inhibiteur peptidaseNM_002638.3 1.19471567 2.5 TCTTGATCGTGGT GACTGGCTCTTGCGC de inhibitor 3, GGTGTTCCTCA TTTGACTTT peptidase skin-derived (SEQ ID(SEQ ID NO: 36) NO: 35) PSME2 Sous unité proteasome NM_002818.21.06606742 1.9 TCCCTCAATGTG TCTCATTCCCAGGGA activa-  activator 2GCTGACTTGACT GAAATCCAC trice du  subunit 2 SEQ ID (SEQ ID NO: 38)proteasome (PA28 beta) (NO: 37) 2 SERPINB3 Membre  serpin NM_006919.20.46222873 4.36633 ACTCCTGGGTGG ACTCCTGGGTGGAA 3 des peptidase 7 6795AAAGTCAAACGA AGTCAAACGA Inhibi-  inhibitor, (SEQ ID (SEQ ID NO: 40)teursde la clade B NO: 39) serpine (ovalbumin), peptidase member 3AKR1B10 Membre B10 aldo-keto NM_020299.4 1.219844195 1.9 AAGATGATAAAGGAGCTTCTCGATCTGG de la  reductase TAATGCCATCG AAGTGGCTG famille family 1,(SEQ ID (SEQ ID NO: 42) des  member B10 NO: 41) aldo-keto (aldosereductases reductase) AKR1C1 Membre C1 aldo-keto NM_001353.5 1.5876814033.432182 GCAGAGGTTCCTA ACCTGCTCCTCATTA de la  reductase 344AAAGTAAAGCTTT TTGTATAAATGA famille family 1, A (SEQ ID NO: 44) des member C1 (SEQ ID aldo-keto NO: 43) reductases AKR1C2 Membre C2aldo-keto NM_001354.4 0.686185206 3.101161 CATTGCATGAGGTCCTTAGCTGTAGCTTA de la  reductase 617 TGCCAGAAGG CTGAAGTCG famille family 1, (SEQ ID (SEQ ID NO: 46) des member C2 NO: 45) aldo-ketoreductases CYP1B1 cytochrome cytochrome NM_000104.3 0.751299588 17.37117ATCAACAAGGACC TCATTTGGGTTGGCC P450,  P450, 803 TGACCAGCAGA CTGAAATCGfamille family 1, (SEQ ID (SEQ ID NO: 48) 1, sous subfamily B, NO : 47)famille B, polypeptide 1 polypep- tide 1 FTH1P Polypeptide ferritin,NM_002032.2 1.032878604 1.942318 CTTTGACCGCGATG TCAGTTTCTCAGCAT lourd heavy 842 ATGTGGCTTT GTTCCCTCT de la polypeptide 1 (SEQ ID(SEQ ID NO: 50) ferritine, NO: 49) 1 FTL Polypeptide ferritin,NM_000146.3 0.681444874 2.940199 TTGGATCTTCATGC AGTCGTGCTTGAGAG Léger light 851 CCTGGGTTCT TGAGCCTTT de la  polypeptide (SEQ ID(SEQ ID NO: 52) ferritine, NO: 51) 1 G6PD glucose-6- glucose-6-NM_000402.3 0.581030262 1.7 GAACCTCATGGTGC TGAGGATAACGCAG phosphatephosphate TGAGATTTGC GCGATGTTGT dehydro- dehydrogenase (SEQ ID(SEQ ID NO: 54) genase NO: 53) GCLM Sous unité glutamate- NM_002061.20.874803304 5.083406 ATGGCCTGTTCAGT TCCCAGTAAGGCTGT modifica- cysteine994 CCTTGGAGTT AAATGCTCC trice  ligase, (SEQ ID (SEQ ID NO: 56) de lamodifier NO: 55) glutamate- subunit cysteine ligase NQO2 NAD(P)H NAD(P)HNM_000904.3 1.170224823 2.8 CACGAAGACAGGA CGGATGCAATTTCAG dehydro-dehydrogenase, GTCAATGGAGA GAGCAAAGC genase des  quinone 2 (SEQ ID(SEQ ID NO: 58) quinone s2 NO: 57) SLC7A11 Membre 11 solute NM_014331.30.502347786 5.088930 TATCCCTGGCATTT TGCCCACAGCTGTAA de la  carrier 603GGACGCTACA TGAGCTTGA famille family 7, (SEQ ID (SEQ ID NO: 60) 7 des  (cationic NO: 59) porteurs amino acid de transporter, molécules y+system) solubles member 11 TXNRD1 thiore- thioredoxin NM_182729.10.725407592 3.707572 GGTCCTCACAGGAT TGCCCAAGTAACGTG doxine  reductase 192 TAAGGCAACA GTCTTTCAC reductase (SEQ ID (SEQ ID NO: 62) 1 NO: 61)UGT1A1 Polypeptide UDP glucurono- NM_000463.2 0.527392251 1.408908ACAGAACTTTCTGT AGCCAGACAGATGC A1 de la syltransferase 304 GCGACGTGGTAGAGCTCAAT famille  1 family, (SEQ ID (SEQ ID NO: 64) des UDP polypeptide NO: 63) glucurono- A1 syltrans- ferase 1 UGT1A9 PolypeptideUDP glucurono- NM_021027.2 0.568212175 1.402794 GCTTTGCCGAGGCAATGAGTTTCTCCACC A9 de la syltransferase 916 GGGAAGCTAC ACCGACCTCfamille  1 family, (SEQ ID (SEQ ID NO: 66) des UDP  polypeptide NO: 65)glucurono- A9 syltrans- ferase 1 YWHAZ Polypeptide tyrosine 3-NM_001135702. 1.154023086 3.1 ACAGCAGATGGCT TGCTCTCTGCTTGTG zeta de lamonooxygenase/ 1 CGAGAATACAG AAGCATTGG proteine tryptophan 5- (SEQ ID(SEQ ID NO: 68) d'activa- monooxygenase NO: 67) tion  activationde tyrosine protein, zeta 3-monooxy-  polypeptide genase/ tryptophan5-monooxy- genase polypeptide CD36 CD36  CD36 molecule NM_001127444.0.640243459 5.375531 ACAGATGCAGCCT GGGATTCCTTTCAGATT molecule(thrombospondin 509 CATTTCCACCT AACGTCGG receptor) (SEQ ID(SEQ ID NO: 70) NO: 69) CYP1A1 cytochrome cytochrome NM_000499.30.549457486 3.763056 AGTGGCAGATCAA ACACCTTGTCGATAGC P450,  P450, family606 CCATGACCAGA ACCATCAG famille 1, subfamily (SEQ ID (SEQ ID NO: 72)1, sous A, polypeptide NO: 71) famille A, 1 poly- peptide 1 GCLCSous unité glutamate- NM_001498.2 0.905634705 2.728685 AATGGGCAATTGCTAAAGGGAGATGCAGCA catalitique cysteine 624 GTCTCCAGGT CTCAAAGC de laligase, (SEQ ID (SEQ ID NO: 74) glutamate- catalytic NO: 73) cysteinesubunit ligase HMOX1 heme heme NM_002133.1 0.76137536 1.461320GGGCCAGCAACAA TCGCCACCAGAAAGCT oxygenase  oxygenase 376 AGTGCAAGATTGAGTGTAA 1 (decycling) (SEQ ID (SEQ ID NO: 76) 1 NO: 75) NQO1 NAD(P)HNAD(P)H NM_001025434. 0.740574662 1.948391 AGTGGCTCCATGTATTCTCCAGGCGTTTCTT dehydro- dehydrogenase, 1 073 CTCTCTGCAA CCATCCTgenase, quinone 1 (SEQ ID (SEQ ID NO: 78) des  NO: 77) quinones 1 S100A8Protéine S100 calcium NM_002964.3 0.970410479 1.890390 GGGATGACCTGAATGTTGATATCCAACTCT de fixation binding 064 GAAATTGCTA TTGAACCA du calciumprotein A8 (SEQ ID (SEQ ID NO: 80) S100 A8 NO: 79)

Moreover, 35 substances classified according to their characteristics(non-irritant (non-IRR), non-sensitizer (NS), irritant (IRR) orsensitizing by using the official classification of Extreme (E), Strong(S), Moderate (M) and Weak (W) were tested at different doses (Table 2).For analysis, the dose permitting the maximum response without inducingtoo much tissue destruction (corrosion) was selected.

Class Name CAS# S Hydroquinone 123-31-9 S 2-aminophenol 95-55-6 E4-Nitrobenzylbromide (electrophile) 100-11-8 S 2,4,6-Trinitrobenzenesulfonic acid 2508-19-2 S p-Phenylenediamine 106-50-3 M Citral 5392-40-5S Dinitrochlorobenzene 97-00-7 M Benzaldehyde 100-52-7 W Resorcinol108-46-3 M Benzisothiazolin-3-one (Benzisothiazolinone) 2634-33-5 MTetramethylthiuram disulfide 137-26-8 S Oxazolone 15646-46-5 SChloroatranol 57074-21-2 E Diphenylcyclopropenone 886-38-4 S PotassiumDichromate 7778-50-9 M cinnamaldehyde 104-55-2 S2-Bromo-2-(bromomethyl)glutaronitrile 35691-65-7 M Glyoxal 107-22-2 MSaccharine 6485-34-3 S Formaldehyde 50-00-0 M Trimellitic anhydride552-30-7 E Methylchloroisothiazolinone 26172-55-4 M Phenyl Acetaldehyde122-78-1 W Benzyl benzoate 120-51-4 W Lilial 80-5406 W alpha-Hexylcinnamaldehyde 101-86-0 W Eugenol 97-53-0 M 2-mercaptobenzothiazole149-30-4 S Glutaraldehyde 111-30-8 M Isoeugenol 97-54-1 NS DipropyleneGlycol 25265-71-8 NS Glycerol 56-81-5 NS Cetyl trimethylammonium bromide57-09-0 NS Limonen 5989-54-8 IRR Lactic acid (cys pos) 598-82-3 IRRSodium lauryl sulfate 151-21-3

Three groups of biomarkers were tested on the samples, those specificfor irritation, those represented by the ARE gene family (genes underthe control of ARE “antioxidant responsive element promoters”, i.e.,AKR1B10, AKR1C1, AKR1C2, CYP1B1, FTH1P, FTL, G6PD, GCLM, NQO2, SLC7A11,TXNRD1, UGT1A1, UGT1A9, YWHAZ, CD36, CYP1A1, GCLC, HMOX1, NQO1, PSME2and S100A8 genes) and another group of genes specific for sensitization,notably for sensitizing substances that do not induce ARE genes.

Irritants for which the expression of the IL-8 gene is 50 times greaterthan the control and that induce a non-specific expression of ARE geneswere retested at lower doses.

The results are presented in Tables 2 and 3, with a code according tothe degree of overexpression of the genes. If the expression is greaterthan 1.3 compared to the control, a grade of 11s given; if thisexpression is lower, the grade is 0. Note that, preferably, if thenumber of ARE genes is greater than 11, one can conclude that thesubstance is certainly sensitizing. For substances that do not stronglyinduce ARE genes, the other group of sensitization genes is analyzed.

Observe that, preferably, if at least 8 of these genes areoverexpressed, then the substance is clearly sensitizing.

Preferably, said test is done both on the group of ARE genes and thegroup of “non-ARE” genes.

TABLE 2

In order to be able to evaluate the sensitizing power of the compounds,a selection of compounds was subjected to successive dilutions in orderto be tested again for their sensitizing potential, as shown in Table 3.

DAPK2 CTSS EHF NGAL S100A8 DEFB1 P13 CXCR1 IL1R2 FABP4 IVL CD36 HSP27methyl-chloro- isothiazolinone MCIT-50% 1.0 2.0 5.8 2.2 1.6 3.0 2.2 1.65.1 1.5 5.0 0.8 1.3 NUMBER OF GENES 0 1 1 1 1 1 1 1 1 1 1 0 1 MCIT-10%0.8 1.1 3.7 2.2 1.5 2.6 2.1 1.0 3.2 1.4 3.7 0.7 1.1 NUMBER OF GENES 0 01 1 1 1 1 0 1 1 1 0 0 MCIT-1% 0.8 1.3 1.8 1.2 1.2 1.7 1.6 1.2 1.7 1.32.2 0.8 0.8 NUMBER OF GENES 0 0 1 0 0 1 1 0 1 1 1 0 0 MCIT-0.1% 0.6 0.71.1 2.2 1.0 1.1 1.9 1.4 1.7 1.0 1.3 1.1 1.3 NUMBER OF GENES 0 0 0 1 0 01 1 1 0 0 0 0 diphenyl cyclopropenone DPCP-50% 1.2 0.9 2.1 0.6 1.1 1.41.2 1.5 1.2 0.9 1.4 1.0 1.1 NUMBER OF GENES 0 0 1 0 0 1 0 1 0 0 1 0 0DPCP-10% 1.2 0.9 1.2 1.2 1.1 1.1 1.1 1.2 0.9 1.0 1.3 1.0 1.0 NUMBER OFGENES 0 0 0 0 0 0 0 0 0 0 0 0 0 DPCP-1% 1.2 1.0 1.2 1.0 1.2 1.3 1.1 1.51.4 0.8 1.3 1.3 1.0 NUMBER OF GENES 0 0 0 0 0 0 0 1 1 0 0 0 0 DPCP-0.1%1.3 1.6 0.8 1.1 1.0 1.2 1.1 2.3 1.1 1.1 1.3 0.8 0.9 NUMBER OF GENES 0 10 0 0 0 0 1 0 0 0 0 0 TOTAL KRT4 BRAK SERPINB3 MELANA HSP90 PDZK1iP1IL18 DHFR SENS GCLC NQO1 FTL methyl-chloro- isothiazolinone MCIT-50% 1.10.3 3.2 0.4 3.5 0.8 0.9 0.6 16.5 4.6 17.0 NUMBER OF 0 0 1 0 1 0 0 0 13 11 1 GENES MCIT-10% 1.6 0.3 2.7 0.6 2.0 0.9 0.7 0.7 14.8 6.8 11.0 NUMBEROF 1 0 1 0 1 0 0 0 11 1 1 1 GENES MCIT-1% 1.2 0.5 1.7 0.7 1.3 0.9 0.80.8 5.3 5.1 4.4 NUMBER OF 0 0 1 0 1 0 0 0 8 1 1 1 GENES MCIT-0.1% 2.41.5 1.6 0.3 1.4 1.3 1.3 1.2 1.7 1.8 1.2 NUMBER OF 1 1 1 0 1 1 0 0 9 1 10 GENES diphenyl cyclopropenone DPCP-50% 0.5 0.3 1.2 1.2 2.7 0.8 0.9 0.97.2 6.2 4.9 NUMBER OF 0 0 0 0 1 0 0 0 5 1 1 1 GENES DPCP-10% 0.7 0.7 1.01.5 1.2 1.0 0.8 0.9 5.0 4.3 3.2 NUMBER OF 0 0 0 1 0 0 0 0 4 1 1 1 GENESDPCP-1% 1.0 0.7 1.3 0.4 1.2 1.2 1.2 0.8 1.7 2.0 1.5 NUMBER OF 0 0 0 0 00 0 0 2 1 1 1 GENES DPCP-0.1% 1.0 1.2 0.9 0.8 1.0 0.9 0.6 0.9 1.0 1.81.4 NUMBER OF 0 0 0 0 0 0 0 0 2 0 1 1 GENES FTH1P AKR1B10 AKR1C2 AKR1C1CYP1B1 CYP1A1 TXNRD1 HMOX1 UGT1A1 UGT1A9 methyl-chloro- isothiazolinoneMCIT-50% 5.7 1.0 10.6 24.8 177.9 3855.0 45.1 22.4 1.3 1.7 NUMBER OF 1 01 1 1 1 1 1 1 1 GENES MCIT-10% 3.8 1.3 9.6 27.0 49.5 852.8 29.7 14.3 1.41.2 NUMBER OF 1 1 1 1 1 1 1 1 1 0 GENES MCIT-1% 2.0 1.8 6.6 15.6 35.0217.3 10.9 3.1 2.1 1.5 NUMBER OF 1 1 1 1 1 1 1 1 1 1 GENES MCIT-0.1% 1.11.4 1.5 1.3 49.0 115.5 1.5 1.5 2.1 1.0 NUMBER OF 0 0 1 0 1 1 1 1 1 0GENES diphenyl cyclopropenone DPCP-50% 2.5 1.9 7.3 19.3 32.5 13.9 23.510.0 3.4 1.9 NUMBER OF 1 1 1 1 1 1 1 1 1 1 GENES DPCP-10% 2.3 2.0 5.09.8 68.6 87.6 10.0 2.2 4.6 3.1 NUMBER OF 1 1 1 1 1 1 1 1 1 1 GENESDPCP-1% 1.3 1.2 2.1 3.6 40.5 45.7 3.7 1.6 2.5 1.8 NUMBER OF 0 0 1 1 1 11 1 1 1 GENES DPCP-0.1% 1.2 1.1 1.6 0.9 2.6 9.2 1.6 0.9 2.1 1.1 NUMBEROF 0 0 1 0 1 1 1 0 1 0 GENES NQO2 SLC7A11 GCLM AHR TOTAL ARE SENS >1.3ARE >1.3 methyl-chloro- isothiazolinone MCIT-50% 2.0 29.1 21.8 1.0NUMBER OF 1 1 1 0 15 13 15 Sensitizing EXTREME GENES MCIT-10% 1.8 26.519.1 1.3 NUMBER OF 1 1 1 1 16 11 16 Sensitizing GENES MCIT-1% 1.6 12.811.3 1.1 NUMBER OF 1 1 1 0 16 8 16 Sensitizing GENES MCIT-0.1% 1.4 2.31.5 1.6 NUMBER OF 0 1 1 1 11 9 11 Sensitizing GENES diphenylcyclopropenone DPCP-50% 1.5 24.3 13.8 1.4 NUMBER OF 1 1 1 1 17 5 17Sensitizing EXTREME GENES DPCP-10% 1.7 13.5 7.3 0.8 NUMBER OF 1 1 1 0 164 16 Sensitizing GENES DPCP-1% 1.4 2.8 2.4 0.6 NUMBER OF 1 1 1 0 14 2 14Sensitizing GENES DPCP-0.1% 1.1 1.7 1.3 0.9 NUMBER OF 0 1 0 0 8 2 8Sensitizing GENES DAPK2 CTSS EHF NGAL S100A8 DEFB1 P13 CXCR1 IL1R2 FABP4IVL CD36 HSP27 2-aminophenol 2-aminophenol- 1.0 1.3 1.7 1.8 1.3 1.9 1.52.3 2.0 1.1 1.8 0.7 1.0 50% NUMBER OF GENES 0 1 1 1 1 1 1 1 1 0 1 0 02-aminophenol- 1.0 1.2 1.5 1.9 1.2 1.8 1.4 1.5 1.8 1.1 1.8 0.5 1.1 10%NUMBER OF GENES 0 0 1 1 0 1 1 1 1 0 1 0 0 2-aminophenol- 0.9 1.4 1.4 1.51.2 1.4 1.2 2.0 1.3 0.7 1.3 0.6 0.8 1% NUMBER OF GENES 0 1 1 1 0 1 0 1 00 1 0 0 2-aminophenol- 1.0 1.4 1.2 1.1 1.4 1.1 1.3 1.0 1.0 0.9 0.9 0.90.8 0.1% NUMBER OF GENES 0 1 0 0 1 0 0 0 0 0 0 0 0 Hydroquinonehydroquinone- 1.0 1.6 1.5 1.3 1.4 2.0 1.7 2.9 1.5 1.4 2.6 0.8 1.3 50%NUMBER OF GENES 0 1 1 0 1 1 1 1 1 1 1 0 0 hydroquinone- 1.2 0.8 1.2 2.01.4 1.5 1.4 2.2 1.4 1.2 1.8 0.7 1.0 10% NUMBER OF GENES 0 0 0 1 0 1 0 10 0 1 0 0 hydroquinone-1% 1.2 1.2 1.1 1.7 1.4 1.1 1.2 2.8 1.2 1.1 1.50.7 0.9 NUMBER OF GENES 0 0 0 1 1 0 0 1 0 0 1 0 0 hydroquinone- 1.1 0.71.2 1.5 1.1 1.7 1.1 2.9 1.3 1.1 1.6 0.7 0.8 0.1% NUMBER OF GENES 0 0 0 10 1 0 1 0 0 1 0 0 TOTAL KRT4 BRAK SERPINB3 MELANA HSP90 PDZK1iP1 IL18DHFR SENS GCLC NQO1 2-aminophenol 2-aminophenol- 1.7 0.3 1.4 0.2 1.5 1.00.9 0.9 4.0 3.3 50% NUMBER OF GENES 1 0 1 0 1 0 0 0 12 1 12-aminophenol- 1.7 0.3 1.2 0.2 1.5 1.0 0.9 0.9 2.3 2.5 10% NUMBER OFGENES 1 0 0 0 1 0 0 0 9 1 1 2-aminophenol- 1.1 0.5 1.2 0.5 1.2 1.0 0.80.9 2.2 1.1 1% NUMBER OF GENES 0 0 0 0 0 0 0 0 6 1 0 2-aminophenol- 1.70.8 1.3 1.1 1.0 1.0 1.1 1.2 1.1 0.9 0.1% NUMBER OF GENES 1 0 1 0 0 0 0 04 0 0 Hydroquinone hydroquinone- 1.1 0.8 1.1 1.6 1.8 1.0 0.9 1.1 2.4 2.850% NUMBER OF GENES 0 0 0 1 1 0 0 0 11 1 1 hydroquinone- 1.2 0.8 1.1 0.91.2 1.0 0.9 0.7 1.5 1.5 10% NUMBER OF GENES 0 0 0 0 0 0 0 0 4 1 1hydroquinone-1% 1.5 0.8 1.0 0.7 1.1 1.0 0.8 0.8 1.3 1.3 NUMBER OF GENES1 0 0 0 0 0 0 0 5 0 0 hydroquinone- 2.0 0.7 1.1 0.7 1.3 1.2 0.8 0.4 1.31.2 0.1% NUMBER OF GENES 1 0 0 0 0 0 0 0 5 0 0 FTL FTH1P AKR1B10 AKR1C2AKR1C1 CYP1B CYP1A1 TXNRD1 HMOX1 UGT1A1 2-aminophenol 2-aminophenol- 3.52.0 1.3 3.3 8.6 37.7 66.5 6.4 1.9 2.1 50% NUMBER OF GENES 1 1 1 1 1 1 11 1 1 2-aminophenol- 2.5 1.5 1.1 3.4 9.3 45.1 122.0 7.6 1.7 1.8 10%NUMBER OF GENES 1 1 0 1 1 1 1 1 1 1 2-aminophenol- 2.1 1.2 0.9 1.2 4.315.6 14.6 4.8 1.4 1.6 1% NUMBER OF GENES 1 0 0 0 1 1 1 1 1 12-aminophenol- 1.0 0.9 0.8 1.1 1.2 5.3 7.1 3.0 1.1 0.9 0.1% NUMBER OFGENES 0 0 0 0 0 1 1 1 0 0 Hydroquinone hydroquinone- 3.5 2.1 1.1 3.7 3.549.5 57.8 7.9 3.5 1.9 50% NUMBER OF GENES 1 1 0 1 1 1 1 1 1 1hydroquinone- 1.9 1.4 1.0 1.8 2.6 17.1 33.2 2.6 1.2 1.5 10% NUMBER OFGENES 1 1 0 1 1 1 1 1 0 1 hydroquinone-1% 1.5 1.3 1.0 1.3 2.0 7.1 15.42.1 1.0 1.4 NUMBER OF GENES 1 0 0 0 1 1 1 1 0 0 hydroquinone- 1.5 1.20.9 0.7 1.2 1.8 8.0 2.3 1.1 1.7 0.1% NUMBER OF GENES 1 0 0 0 0 1 1 1 0 1TOTAL UGT1A9 NQO2 SLC7A11 GCLM AHR ARE SENS >1.3 ARE >1.3 2-aminophenol2-aminophenol- 0.4 1.3 6.0 4.7 1.3 50% NUMBER OF GENES 0 0 1 1 0 14 1214 Sensitizing STRONG 2-aminophenol- 1.2 1.4 3.8 4.6 0.7 10% NUMBER OFGENES 0 1 1 1 0 14 9 14 Sensitizing 2-aminophenol- 1.0 1.1 3.2 3.5 0.61% NUMBER OF GENES 0 0 1 1 0 10 6 10 Sensitizing 2-aminophenol- 1.1 1.02.1 2.1 1.0 0.1% NUMBER OF GENES 0 0 1 1 0 5 4 5 Non- sensitizingHydroquinone hydroquinone- 1.7 2.0 5.4 3.8 0.9 50% NUMBER OF GENES 1 1 11 0 15 11 15 Sensitizing STRONG hydroquinone- 1.3 1.5 1.6 2.0 0.6 10%NUMBER OF GENES 0 1 1 1 0 13 4 13 Sensitizing hydroquinone-1% 1.5 1.41.5 1.8 0.6 NUMBER OF GENES 1 1 1 1 0 9 5 9 Sensitizing hydroquinone-0.8 0.9 1.3 0.7 0.8 0.1% NUMBER OF GENES 0 0 0 0 0 5 2 5 Non-sensitizing DAPK2 CTSS EHF NGAL S100A8 DEFB1 P13 CXCR1 IL1R2 FABP4 IVLCD36 HSP27 Isoeugenol isoeugenol-50% 1.4 1.4 2.0 2.7 1.5 2.1 1.7 2.6 1.71.8 2.8 0.6 1.2 NUMBER OF GENES 1 1 1 1 1 1 1 1 1 1 1 0 0 isoeugenol-10%0.9 1.5 1.4 2.1 1.3 1.5 1.4 1.6 1.4 1.2 1.9 0.6 0.9 NUMBER OF GENES 0 11 1 1 1 1 1 1 0 1 0 0 isoeugenol-1% 0.9 0.8 1.2 1.1 1.0 1.1 0.9 1.1 1.40.9 1.4 1.3 0.8 NUMBER OF GENES 0 0 0 0 0 0 0 0 1 0 1 1 0isoeugenol-0.1% 1.1 0.9 1.5 1.1 1.1 1.0 1.1 0.7 1.2 0.8 1.3 0.9 1.0NUMBER OF GENES 0 0 1 0 0 0 0 0 0 0 1 0 0 Glyoxal Glyoxal-50% 0.9 2.70.9 0.6 0.9 1.6 1.4 2.7 2.0 0.8 1.5 1.1 0.8 NUMBER OF GENES 0 1 0 0 0 11 1 1 0 1 0 0 Glyoxal-10% 0.9 2.2 0.9 1.3 0.9 1.6 1.3 1.8 2.2 0.8 1.61.3 0.8 NUMBER OF GENES 0 1 0 1 0 1 1 1 1 0 1 1 0 Glyoxal-1% 0.9 1.3 0.90.6 0.9 1.4 1.4 1.6 1.4 0.8 1.7 1.1 0.8 NUMBER OF GENES 0 1 0 0 0 1 1 11 0 1 0 0 Glyoxal-0.1% 0.9 1.0 0.8 0.7 0.7 0.8 0.8 1.1 0.9 0.4 0.7 1.10.7 NUMBER OF GENES 0 0 0 0 0 0 0 0 0 0 0 0 0 TOTAL KRT4 BRAK SERPINB3MELANA HSP90 PDZK1iP1 IL18 DHFR SENS GCLC NQO1 Isoeugenol isoeugenol-50%1.2 0.6 1.5 0.4 1.7 1.1 0.8 0.9 1.3 1.8 NUMBER OF GENES 0 0 1 0 1 0 0 013 1 1 isoeugenol-10% 1.0 0.8 1.1 0.6 1.3 1.0 0.7 0.9 0.8 1.0 NUMBER OFGENES 0 0 0 0 1 0 0 0 10 0 0 isoeugenol-1% 0.8 0.9 1.2 1.4 1.3 1.1 1.10.9 0.9 1.0 NUMBER OF GENES 0 0 0 1 1 0 0 0 5 0 0 isoeugenol-0.1% 1.20.6 1.2 0.9 0.9 1.0 0.6 0.8 0.8 1.1 NUMBER OF GENES 0 0 0 0 0 0 0 0 2 00 Glyoxal Glyoxal- 0.6 0.5 1.7 1.1 2.0 1.0 1.0 0.9 0.9 0.9 50% NUMBER 00 1 0 1 0 0 0 8 0 0 OF GENES Glyoxal- 1.4 0.5 1.3 1.1 1.5 1.4 1.3 0.91.1 1.2 10% NUMBER 1 0 1 0 1 1 1 0 13 0 0 OF GENES Glyoxal- 0.6 0.5 1.11.1 1.1 1.0 1.0 0.9 1.1 1.1 1% NUMBER 0 0 0 0 0 0 0 0 6 0 0 OF GENESGlyoxal- 0.9 0.6 1.0 0.5 1.0 0.9 0.8 0.9 1.0 1.0 0.1% NUMBER 0 0 0 0 0 00 0 0 0 0 OF GENES FTL FTH1P AKR1B10 AKR1C2 AKR1C1 CYP1B1 CYP1A1 TXNRD1HMOX1 UGT1A1 Isoeugenol isoeugenol-50% 1.5 1.0 1.0 1.3 2.2 39.1 87.1 2.61.4 1.2 NUMBER OF GENES 1 0 0 1 1 1 1 1 1 0 isoeugenol-10% 0.9 0.9 0.50.7 1.1 39.8 510.5 1.5 1.2 1.2 NUMBER OF GENES 0 0 0 0 0 1 1 1 0 0isoeugenol-1% 1.0 0.9 0.7 0.9 1.2 25.3 219.2 1.3 1.2 1.1 NUMBER OF GENES0 0 0 0 0 1 1 1 0 0 isoeugenol-0.1% 1.0 0.8 0.9 1.0 1.1 12.2 11.9 1.10.9 1.0 NUMBER OF GENES 0 0 0 0 0 1 1 0 0 0 Glyoxal Glyoxal- 0.9 0.9 0.80.9 0.8 3.3 1.1 1.4 1.4 1.4 50% NUMBER 0 0 0 0 0 1 0 1 1 1 OF GENESGlyoxal- 1.1 1.1 0.7 1.2 1.0 0.8 0.0 1.3 1.0 1.0 10% NUMBER 0 0 0 0 0 00 1 0 0 OF GENES Glyoxal- 1.1 1.0 1.1 1.0 1.0 1.1 0.9 0.9 0.9 1.2 1%NUMBER 0 0 0 0 0 0 0 0 0 0 OF GENES Glyoxal- 1.0 0.9 0.6 0.8 0.8 0.6 0.61.0 1.0 0.9 0.1% NUMBER 0 0 0 0 0 0 0 0 0 0 OF GENES TOTAL ARE UGT1A9NQO2 SLC7A11 GCLM AHR ARE SENS >1.3 >1.3 Isoeugenol isoeugenol-50% 1.21.1 2.2 2.0 1.0 NUMBER OF GENES 0 0 1 1 0 11 13 11 Sensitizing MODERATEisoeugenol-10% 1.6 0.9 1.1 1.5 0.9 NUMBER OF GENES 1 0 0 1 0 5 10 5Sensitizing isoeugenol-1% 1.0 1.0 1.1 1.2 0.9 NUMBER OF GENES 0 0 0 0 03 5 3 Sensitizing isoeugenol-0.1% 0.8 1.0 1.1 1.1 0.7 NUMBER OF GENES 00 0 0 0 2 2 2 Sensitizing Glyoxal Glyoxal- 1.3 0.9 1.0 1.5 0.9 50%NUMBER 1 0 0 1 0 6 8 6 Sensitizing MODERATE OF GENES Glyoxal- 0.2 1.01.9 1.7 1.0 10% NUMBER 0 0 1 1 0 3 13 3 Sensitizing OF GENES Glyoxal-0.6 1.0 1.4 0.9 1.1 1% NUMBER 0 0 1 0 0 1 6 1 Sensitizing OF GENESGlyoxal- 0.3 0.8 1.0 0.9 0.9 0.1% NUMBER 0 0 0 0 0 0 0 0 Sensitizing OFGENES DAPK2 CTSS EHF NGAL S100A8 DEFB1 P13 CXCR1 IL1R2 FABP4 IVL CD36HSP27 Eugenol Eugenol-50% 1.5 0.1 1.2 1.3 1.9 3.0 1.4 2.2 1.3 1.4 2.62.3 5.4 NUMBER OF GENES 1 0 0 1 1 1 1 1 1 1 1 1 1 Eugenol-10% 1.3 1.01.3 0.7 1.0 1.1 0.9 0.8 0.8 0.6 0.9 1.7 1.1 NUMBER OF GENES 1 0 1 0 0 00 0 0 0 0 1 0 alpha hexyl cinnamaldehyde HCA-50% 0.8 0.8 1.3 1.1 1.1 1.11.0 1.2 1.1 1.2 1.4 1.9 1.1 NUMBER OF GENES 0 0 1 0 0 0 0 0 0 0 1 1 0HCA-10% 1.0 1.0 1.0 0.8 1.0 0.9 0.9 0.7 0.8 0.8 0.9 2.2 1.0 NUMBER OFGENES 0 0 0 0 0 0 0 0 0 0 0 0 0 ipropylene glycol DPG-50% 1.3 1.2 0.91.0 1.2 1.2 1.0 2.2 1.2 1.2 1.1 1.1 1.1 NUMBER OF GENES 1 0 0 0 0 0 0 10 0 0 0 0 DPG-10% 1.4 0.7 1.0 0.6 1.1 0.9 1.0 0.5 1.0 0.8 0.9 1.1 1.2KRT4 BRAK SERPINB3 MELANA HSP90 PDZK1iP1 IL18 DHFR TOTAL SENS EugenolEugenol-50% 4.6 0.4 1.4 1.0 9.1 1.7 0.7 0.6 NUMBER OF GENES 1 0 1 0 1 10 0 15 Eugenol-10% 0.8 0.7 1.2 1.9 1.1 0.9 1.1 0.8 NUMBER OF GENES 0 0 01 0 0 0 0 4 alpha hexyl cinnamaldehyde HCA-50% 1.0 0.8 1.2 1.3 1.7 1.11.0 0.9 NUMBER OF GENES 0 0 0 1 1 0 0 0 5 HCA-10% 0.7 1.0 1.5 2.0 1.61.1 1.1 0.8 NUMBER OF GENES 0 0 0 1 1 0 0 0 2 ipropylene glycol DPG-50%1.3 1.1 1.1 0.6 1.0 1.1 1.2 1.2 NUMBER OF GENES 1 0 0 0 0 0 0 0 3DPG-10% 1.1 0.8 1.2 0.6 0.9 1.0 1.1 1.1 GCLC NQO1 FTL FTH1P AKR1B10AKR1C2 AKR1C1 CYP1B1 CYP1A1 TXNRD1 HMOX1 Eugenol Eugenol-50% 3.2 0.7 4.22.3 1.0 0.7 0.7 0.9 2.3 3.0 2.4 NBRE GENES 1 1 0 1 1 0 0 0 0 1 1Eugenol-10% 1.3 1.1 1.1 1.1 0.9 1.1 1.3 0.6 0.5 1.1 1.3 NBRE GENES 1 0 00 0 0 1 0 0 0 1 alpha hexyl cinnamaldehyde HCA-50% 0.9 1.5 1.7 1.0 1.11.2 1.1 1.7 12.3 1.5 1.0 NBRE GENES 0 1 1 0 0 1 0 1 1 1 0 HCA-10% 1.01.2 1.1 1.1 1.1 1.1 0.9 0.8 0.3 0.9 1.1 NBRE GENES 0 0 0 0 0 0 0 0 0 0 0Dipropylene glycol DPG-50% 1.0 1.2 1.2 1.2 1.1 1.4 1.2 1.6 1.5 1.2 1.1NBRE GENES 0 0 0 0 0 1 0 1 1 0 0 DPG-10% 1.1 1.0 1.0 1.1 1.0 0.9 1.3 0.40.2 1.1 1.1 NBRE GENES 0 0 0 0 0 1 0 0 0 0 0 TOTAL ARE UGT1A1 UGT1A9NQO2 SLC7A11 GCLM AHR ARE SENS >1.3 >1.3 Eugenol Eugenol-50% 0.5 25.01.3 2.6 1.1 1.3 NBRE GENES 1 1 1 1 0 1 10 15 10 Sensitizing WEAKEugenol-10% 1.0 0.7 1.0 1.2 1.1 0.8 NBRE GENES 1 0 0 0 0 0 3 4 3Non-sensitizing alpha hexyl cinnamaldehyde HCA-50% 0.8 1.6 1.2 1.3 1.21.0 NBRE GENES 0 1 0 1 0 0 8 5 8 Sensitizing WEAK HCA-10% 0.7 0.7 1.10.8 0.8 0.8 NBRE GENES 0 0 0 0 0 0 0 2 0 Non-sensitizing Dipropyleneglycol DPG-50% 1.3 1.2 1.3 0.9 1.0 0.9 NBRE GENES 0 0 1 0 0 0 4 3 4Non-sensitizing DPG-10% 1.2 0.4 1.0 1.1 1.0 0.9 NBRE GENES 0 0 0 0 0 0 11 1 DAPK2 CTSS EHF NGAL S100A8 DEFB1 P13 CXCR1 IL1R2 FABP4 IVL CD36HSP27 Acide Lactique Lactic acid 1.1 1.4 0.9 1.0 1.1 1.1 0.9 0.8 0.8 0.80.9 0.9 0.8 50% NUMBER OF GENES 0 1 0 0 0 0 0 0 0 0 0 0 0 Lactic acid1.1 1.1 0.9 1.0 1.0 1.0 0.8 0.7 1.0 0.9 0.9 1.1 0.9 10% NUMBER OF GENES0 0 0 0 0 0 0 0 0 0 0 0 0 Lactic acid 1.2 1.0 0.9 0.9 0.8 0.9 0.8 0.71.0 0.7 1.0 1.1 1.0 1% NUMBER OF GENES 0 0 0 0 0 0 0 0 0 0 0 0 0 SLSSLS-50% 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (Corrosive)NUMBER OF GENES 0 0 0 0 0 0 0 0 0 0 0 0 0 SLS-10% 1.2 1.4 0.4 0.9 1.20.8 0.4 1.0 0.8 1.1 1.0 1.1 0.3 NUMBER OF GENES 0 1 0 0 0 0 0 0 0 0 0 00 SLS-1% 0.9 1.4 1.2 1.1 0.9 1.1 1.1 0.6 0.7 1.2 1.1 0.6 1.1 NUMBER OFGENES 0 1 0 0 0 0 0 0 0 0 0 0 0 KRT4 BRAK SERPINB3 MELANA HSP90 PDZK1iP1IL18 DHFR TOTAL SENS Acide Lactique Lactic acid 0.8 0.7 1.2 0.6 0.9 1.00.8 0.9 50% NUMBER OF GENES 0 0 0 0 0 0 0 0 1 Lactic acid 0.8 1.0 1.10.8 0.9 1.0 1.0 1.0 10% NUMBER OF GENES 0 0 0 0 0 0 0 0 0 Lactic acid0.9 0.8 0.9 0.7 1.0 1.0 0.9 0.9 1% NUMBER OF GENES 0 0 0 0 0 0 0 0 0 SLSSLS-50% 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (Corrosive) NUMBER OF GENES 0 00 0 0 0 0 0 0 SLS-10% 0.7 1.4 0.7 2.5 0.7 0.6 1.0 1.2 NUMBER OF GENES 01 0 1 0 0 0 0 3 SLS-1% 1.1 1.3 1.1 1.6 0.6 0.7 0.5 0.8 NUMBER OF GENES 00 0 1 0 0 0 0 2 GCLC NQO1 FTL FTH1P AKR1B10 AKR1C2 AKR1C1 CYP1B1 CYP1A1TXNRD1 HMOX1 Acide Lactique Ac Lactique- 0.9 1.0 1.1 0.9 1.0 0.8 1.2 1.01.1 1.0 1.0 50% NUMBER OF 0 0 0 0 0 0 0 0 0 0 0 GENES Ac Lactique- 0.91.0 1.0 0.9 1.0 1.0 1.2 1.3 1.8 1.2 1.1 10% NUMBER OF 0 0 0 0 0 0 0 0 10 0 GENES Ac Lactique- 1.0 0.9 1.2 1.1 0.7 1.0 1.0 1.1 0.6 1.0 0.9 1%NUMBER OF 0 0 0 0 0 0 0 0 0 0 0 GENES SLS SLS-50% 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 (Corrosif) NUMBER OF 0 0 0 0 0 0 0 0 0 0 0 GENESSLS-10% 1.7 1.1 1.6 1.3 1.1 1.2 1.6 0.3 0.2 1.2 0.5 NUMBER OF 1 0 1 0 00 1 0 0 0 0 GENES SLS-1% 1.3 1.1 1.2 1.0 1.0 1.1 1.0 0.6 1.0 0.9 0.4NUMBER OF 0 0 0 0 0 0 0 0 0 0 0 GENES UGT1A1 UGT1A9 NQO2 SLC7A11 GCLMAHR TOTAL ARE SENS >1.3 ARE >1.3 Acide Lactique Ac Lactique- 1.3 0.8 1.21.0 1.1 0.8 50% NUMBER OF 0 0 0 0 0 0 0 1 0 Non-sensitizing GENES AcLactique- 1.3 0.7 1.1 1.2 1.0 0.5 10% NUMBER OF 0 0 0 0 0 0 1 0 1 GENESAc Lactique- 0.8 0.4 0.9 1.0 0.8 0.9 1% NUMBER OF 0 0 0 0 0 0 0 0 0GENES SLS SLS-50% 0.0 0.0 0.0 0.0 0.0 0.0 (Corrosif) NUMBER OF 0 0 0 0 00 0 0 0 GENES SLS-10% 1.0 1.4 1.0 5.2 1.0 1.4 NUMBER OF 0 0 0 1 0 1 5 35 Non- GENES sensitizing SLS-1% 0.5 0.6 1.1 2.9 1.0 1.2 NUMBER OF 0 0 01 0 0 1 2 1 GENES

1. A method for evaluating the sensitizing potential of a test compound,comprising the steps of: a) contacting a test compound with a biologicalsample; b) determining the expression level of at least six genes chosenin the group consisting of: BRAK (CXC chemokine ligand 14), CTSS(cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4 (fattyacid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18(IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolatereductase), EHF (ets homologous factor), IVL (involucrin) KRT4 (keratin4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1 interactingprotein interaction 1), PI3 (peptidase inhibitor 3), PSME2 (proteasomeactivator subunit 2), SERPINB3 (serpin peptidase inhibitor member 3),AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1 (aldo-ketoreductase family 1, member C1), AKR1C2 (aldo-keto reductase family 1,member C2), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin, lightpolypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM(glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)Hdehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11),TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family,polypeptide A9), YWHAZ (tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein, zeta polypeptide), CD36 (CD36molecule), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide1), GCLC (glutamate-cysteine ligase, catalytic subunit), HMOX1 (hemeoxygenase 1), NQO1 (NAD(P)H dehydrogenase, quinone 1) and S100A8 (S100calcium binding protein A8).
 2. The evaluation method according claim 1,further comprising a step of comparing the expression level of each ofsaid at least six genes with a reference value.
 3. The evaluation methodaccording to claim 1, in which the expression level of each of said atleast six genes is determined by measuring the expression level of thepolypeptide encoded by said gene or a fragment thereof, or bydetermining the expression level of the mRNA from said at least one geneor a fragment thereof.
 4. The evaluation method according to claim 1,comprising an additional step of amplifying the mRNA or cDNA of each ofsaid six genes, the complementary sequence thereof or a fragmentthereof.
 5. The method according to claim 1, characterized in that thebiological sample is a skin sample, preferably a skin samplereconstructed in vitro.
 6. The method according to claim 1, in whichstep b) comprises measuring the expression of at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24 and morepreferentially, at least 25 genes chosen in the group consisting of:BRAK (CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2(death-associated protein kinase 2), FABP4 (fatty acid binding protein4), HSP27 (heat shock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock90 kDa protein), IL1R2 (interleukin-1 receptor type II), TPSAB1(tryptase alpha/beta 1), CXCR1 (interleukin 8 receptor, alpha), DEFB1(defensin, beta 1), DHFR (dihydrofolate reductase), EHF (ets homologousfactor), IVL (involucrin) KRT4 (keratin 4), MELANA (melan-A), NGAL(lipocalin 2), PDZK1IP1 (PDZK1 interacting protein interaction 1), PI3(peptidase inhibitor 3), PSME2 (proteasome activator subunit 2),SERPINB3 (serpin peptidase inhibitor member 3), AKR1B10 (aldo-ketoreductase family 1, member B10), AKR1C1 (aldo-keto reductase family 1,member C1), AKR1C2 (aldo-keto reductase family 1, member C2), CYP1B1(cytochrome P450, family 1, subfamily B, polypeptide 1), FTH1P(ferritin, heavy polypeptide 1), FTL (ferritin, light polypeptide 1),G6PD (glucose-6-phosphate dehydrogenase), GCLM (glutamate-cysteineligase modifier subunit), NQO2 (NAD(P)H dehydrogenase, quinone 2),SLC7A11 (solute carrier family 7, Member 11), TXNRD1 (thioredoxinreductase 1), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptideA1), UGT1A9 (UDP glucuronosyltransferase 1 family, polypeptide A9),YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activationprotein, zeta polypeptide), CD36 (CD36 molecule), CYP1A1 (cytochromeP450, family 1, subfamily A, polypeptide 1), GCLC (glutamate-cysteineligase, catalytic subunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)Hdehydrogenase, quinone 1) and S100A8 (S 100 calcium binding protein A8).7. The method according to claim 1, in which step b) is done between 2and 24 hours after step a), still more preferably between 4 and 18 hoursafter step a), particularly preferentially between 5 and 7 hours afterstep a) and most preferably of all, 6 hours after step a).
 8. A methodfor evaluating the sensitizing power of a test compound, comprising thefollowing steps: 1) obtaining at least one test compound dilution, and2) determining the sensitizing potential of said test compound at saidat least one dilution by a method such as defined according to claim 1.9. A use of at least one gene chosen from the group consisting of: BRAK(CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associatedprotein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heatshock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein),IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1),CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR(dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin)KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1interacting protein interaction 1), PI3 (peptidase inhibitor 3), PSME2(proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitormember 3), AKR1B10 (aldo-keto reductase family 1, member B10), AKR1C1(aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductasefamily 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B,polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin,light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM(glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)Hdehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11),TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family,polypeptide A9 and YWHAZ (tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein, zeta polypeptide) for the in vitroevaluation of the sensitizing potential of a test compound.
 10. A kitfor the implementation of a method for in vitro evaluation of thesensitizing potential of a test compound according to claim 1,comprising means for determining the expression level of at least sixgenes selected the group consisting of BRAK (CXC chemokine ligand 14),CTSS (cathepsin S), DAPK2 (death-associated protein kinase 2), FABP4(fatty acid binding protein 4), HSP27 (heat shock 27 kDa protein), IL18(IL-18), HSP90 (heat shock 90 kDa protein), IL1R2 (interleukin-1receptor type II), TPSAB1 (tryptase alpha/beta 1), CXCR1 (interleukin 8receptor, alpha), DEFB1 (defensin, beta 1), DHFR (dihydrofolatereductase), EHF (ets homologous factor), WL (involucrin) KRT4 (keratin4), MELANA (melan-A), NGAL (gelatinase associated lipocalin), PDZK1IP1(PDZK1 interacting protein interaction 1), PI3 (peptidase inhibitor 3),PSME2 (proteasome activator subunit 2), SERPINB3 (serpin peptidaseinhibitor member 3), AKR1B10 (aldo-keto reductase family 1, member B10),AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 (aldo-ketoreductase family 1, member C2), CYP1B1 (cytochrome P450, family 1,subfamily B, polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL(ferritin, light polypeptide 1), G6PD (glucose-6-phosphatedehydrogenase), GCLM (glutamate-cysteine ligase modifier subunit), NQO2(NAD(P)H dehydrogenase, quinone 2), SLC7A11 (solute carrier family 7,Member 11), TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDPglucuronosyltransferase 1 family, polypeptide A1), UGT1A9 (UDPglucuronosyltransferase 1 family, polypeptide A9), YWHAZ (tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, zetapolypeptide), CD36 (CD36 molecule), CYP1A1 (cytochrome P450, family 1,subfamily A, polypeptide 1), GCLC (glutamate-cysteine ligase, catalyticsubunit), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H dehydrogenase,quinone 1) and S100A8 (S100 calcium binding protein A8).
 11. The kitaccording to claim 10, comprising at least six primer pairs eachamplifying at least one gene chosen from the group consisting of: BRAK(CXC chemokine ligand 14), CTSS (cathepsin S), DAPK2 (death-associatedprotein kinase 2), FABP4 (fatty acid binding protein 4), HSP27 (heatshock 27 kDa protein), IL18 (IL-18), HSP90 (heat shock 90 kDa protein),IL1R2 (interleukin-1 receptor type II), TPSAB1 (tryptase alpha/beta 1),CXCR1 (interleukin 8 receptor, alpha), DEFB1 (defensin, beta 1), DHFR(dihydrofolate reductase), EHF (ets homologous factor), IVL (involucrin)KRT4 (keratin 4), MELANA (melan-A), NGAL (lipocalin 2), PDZK1IP1 (PDZK1interacting protein interaction 1), P13 (peptidase inhibitor 3). PSME2(proteasome activator subunit 2), SERPINB3 (serpin peptidase inhibitormember 3), AKR1B10 (aldo-keto reductase family 1, member B10)₁ AKR1C1(aldo-keto reductase family 1, member C1), AKR1C2 (aldo-keto reductasefamily 1, member C2), CYP1B1 (cytochrome P450, family 1, subfamily B,polypeptide 1), FTH1P (ferritin, heavy polypeptide 1), FTL (ferritin,light polypeptide 1), G6PD (glucose-6-phosphate dehydrogenase), GCLM(glutamate-cysteine ligase modifier subunit), NQO2 (NAD(P)Hdehydrogenase, quinone 2), SLC7A11 (solute carrier family 7, Member 11),TXNRD1 (thioredoxin reductase 1), UGT1A1 (UDP glucuronosyltransferase 1family, polypeptide A1), UGT1A9 (UDP glucuronosyltransferase 1 family,polypeptide A9 and YWHAZ (tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein, zeta polypeptide).